Escherichia coli K1 is a leading cause of septicemia and meningitis in newborn infants. Frequent ineffectiveness of conventional antibiotic therapy indicates the need for better understanding of the genetic determinants underlying K1 colonization and invasion, which might suggest new modes of prophylaxis or treatment. Currently, little is known about how K1 colonizes and causes invasive disease, or about the specific mechanisms allowing transition from mucosal surface colonization to penetration of and growth in normally sterile host compartments. However, it is known that the closely related laboratory strain E. coli K-12, is unable to persist in either the intestinal tract or the bloodstream. Thus information on the genetic determinants of colonization and invasion is contained in the differences between the closely related genomes of these two bacteria. In this project, a new method of integrated genetic and physical mapping (comparative macrorestriction mapping with rare- restriction-sites insertions), which exploits genetic map conservation between strains in order to detect chromosomal physical-distance discrepancies between them is used to isolate RS2I8-specific chromosomal segments containing RS218 virulence genes (i.e., pathogenicity islands). First, new tools (vectors for introduction of minitransposons carrying ultra rare restriction sites) for accurate/general implementation of this method will be constructed (specific aim 1). Second, these tools will be used to construct a set of insertions circumscribing the chromosome of K- 12 strain MG1655 (specific aim 2). Third, artificial macrorestriction fragments from MG1655 and RS218 double-insertion mutants (each pair of double mutants containing the identical pair of insertions) will be used to detect unique chromosomal segments from strain RS218 (and from strain K-12) (specific aim 3). Finally, the unique segments will be analyzed for their possible contributions to RS218 virulence (specific aim 4). This new integrated physical and genetic approach allows rapid and efficient isolation of pathogenicity islands for more focused physical and mutational studies. These studies are expected to lead to new multi locus probes for effective epidemilogic surveillance and prevention of E. coli Kl newborn disease, and to improved understanding and possibly more effective treatment of this disease.